Method for removing geniposide or genipin or both

ABSTRACT

An object of the present invention is to provide a method for removing geniposide or genipin or both from a material containing geniposide or genipin or both. The present invention is a method for removing geniposide or genipin or both from a material containing geniposide or genipin or both, the method comprising treating the material containing geniposide or genipin or both using an activated carbon having (a) a methylene blue adsorption ability of 50 ml/g or more; and (b) an iodine adsorption ability of 750 mg/g or more, thereby removing geniposide or genipin or both.

TECHNICAL FIELD

The present invention relates to a method for removing geniposide orgenipin or both.

BACKGROUND ART

Gardenia-derived edible dyes: gardenia blue, gardenia red, and gardeniayellow have hitherto been used.

“Gardenia blue” is generally obtained by subjecting an iridoid glucoside(whose major component is geniposide), obtained by extraction fromgardenia fruits (Gardenia augusta Merrill var. grandiflora Hort.,Gardenia jasminoides Ellis) of the family Rubiaceae, to a β-glucosidasetreatment in the presence of a proteolytic product or the like.Geniposide is converted into genipin (aglycone) by a β-glucosidasetreatment, and genipin is reacted with a proteolytic product or thelike, thereby generating a gardenia blue colorant component (coloringprinciple).

Therefore, generally, “gardenia blue” is not a single compound, but amixture. “Gardenia blue” generally contains raw material-derivedunreacted geniposide or genipin or both. To the best of the Applicant'sknowledge, a method for highly removing geniposide and genipin fromgardenia blue has not been established; therefore, geniposide or genipinor both are contained even in purified gardenia blue.

“Gardenia red” is generally obtained by subjecting an ester hydrolysate(whose major component is geniposidic acid, which is a hydrolysate ofgeniposide) of an iridoid glucoside (whose major component isgeniposide), obtained by extraction from gardenia fruits of the familyRubiaceae, to a β-glucosidase treatment in the presence of a proteolyticproduct.

Therefore, generally, “gardenia red” is not a single compound, but amixture. “Gardenia red” generally contains raw material-derivedunreacted geniposide, genipin generated from the unreacted geniposide,or both. To the best of the Applicant's knowledge, a method for highlyremoving geniposide and genipin from gardenia red has not beenestablished; therefore, geniposide or genipin or both are contained evenin purified gardenia red.

“Gardenia yellow” is generally obtained by aqueous extraction or hydrousethanol extraction of gardenia fruits of the family Rubiaceae. The majorcolorant components of “gardenia yellow” are crocin and crocetin.

Therefore, “gardenia yellow” is also not a single compound, andgenerally contains raw material-derived geniposide.

Genipap (Genipa americana L.) juice (also known as huito juice) or itsextract, which has been widely used for beverages and food, alsocontains genipin.

As in these examples, foods and food additives containing geniposide orgenipin have hitherto been used without major problems.

However, Non-patent Document 1 discloses that oral administration of alarge amount of geniposide to rat induced hepatotoxicity, and that theexpression of the toxicity may be relevant to genipin generated byintestinal bacteria β-glucosidase in rat.

Further, intake of food or beverage containing genipin such as genipapjuice poses a problem such that the genipin bonds to a protein in theskin around the mouth, thereby turning the skin around the mouth blue.

Regarding geniposide removal, Patent Document 1 discloses a technique ofselectively adsorbing crocin, which is a colorant component of gardeniayellow, to a specific synthetic adsorption resin; this thereby removesan iridoid glucoside containing, as a major component, geniposide, whichcauses discoloration of gardenia yellow into green.

Patent Document 1 further discloses that it is not possible to separategeniposide from the colorant component of gardenia yellow by anactivated carbon treatment, since activated carbon non-selectivelyadsorbs both the colorant component of gardenia yellow and geniposide.

CITATION LIST Patent Documents Patent Document 1: JPS57-151657ANon-Patent Documents Non-patent Document 1: Yamano, T. et al., Food ChemToxicol., 28, p. 515-519 (1990) SUMMARY OF INVENTION Technical Problem

Gardenia-derived colorants, genipap juice, and genipap extract havehitherto been generally used; it is not known that a small amount ofgeniposide or genipin contained in them have negative effects on humans.However, considering the circumstances described above, if a method forremoving geniposide or genipin or both from a composition containinggeniposide or genipin or both is provided, such a method would beconsidered useful.

However, there are only few examples of a technique of removinggeniposide or genipin or both from a composition containing geniposideor genipin or both. To the best of the Applicant's knowledge, thetechnique disclosed in Patent Document 1 is the only example.

The principle of the technique disclosed in Patent Document 1 isselective adsorption of crocin, which is the colorant component ofgardenia yellow; this technique can only be used for gardenia yellow.

Therefore, an object of the present invention is to provide a new methodfor removing geniposide or genipin or both from a composition containinggeniposide or genipin or both.

Solution to Problem

In order to attain the above object, the inventors of the presentinvention attempted removing geniposide or genipin or both from acomposition containing geniposide or genipin or both by using variousmethods commonly used as colorant purification methods; however, atfirst, the inventors could not find an effective method (in thisspecification, Reference Examples 1 and 2 show test results of a resintreatment, acid precipitation, and base precipitation).

However, the inventors of the present invention conducted extensiveresearch, and found that by treating a composition containing geniposideor genipin or both using a specific activated carbon, more specifically,an activated carbon having (a) a methylene blue adsorption ability of 50ml/g or more, and (b) an iodine adsorption ability of 750 mg/g or more,it is possible to remove geniposide or genipin or both from thecomposition while preventing the loss of useful components such as thecolorant component of gardenia blue and the colorant component ofgardenia yellow. This is a very surprising discovery because, asdescribed above, it was generally understood to be impossible toseparate geniposide from the colorant component of gardenia yellow by anactivated carbon treatment (Patent Document 1).

The inventors conducted further research based on this finding, andcompleted the present invention.

The present invention includes the following aspects.

Item 1. A method for removing geniposide or genipin or both from amaterial containing geniposide or genipin or both,

the method comprising treating the material containing geniposide orgenipin or both using an activated carbon having:

(a) a methylene blue adsorption ability of 50 ml/g or more; and

(b) an iodine adsorption ability of 750 mg/g or more, thereby removinggeniposide or genipin or both.

Item 2. A method for producing a gardenia-derived colorant in which thetotal content of geniposide and genipin is reduced,

the method comprising the step of removing geniposide or genipin or bothfrom a gardenia-derived colorant containing geniposide or genipin orboth using the method of Item 1.

Item 3. The method according to Item 2, wherein the gardenia-derivedcolorant is gardenia blue, gardenia red, or gardenia yellow.Item 4. A colorant preparation comprising the gardenia-derived colorantof Item 2 or 3.Item 5. A color composition comprising the gardenia-derived colorant ofItem 2 or 3.Item 6. A method for producing a genipap juice or a genipap extract inwhich the total content of geniposide and genipin is reduced,

the method comprising the step of removing genipin from a genipap juiceor a genipap extract containing genipin using the method of item 1.

Item 7. A gardenia blue-containing composition having a total content ofgeniposide and genipin of 300 ppm or less based on a color value of 100.

Advantageous Effects of Invention

The present invention makes it possible to remove geniposide or genipinor both from a material containing geniposide or genipin or both.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing geniposide content based on a color value of100 relative to activated carbon treatment time (Test Example 3,Activated carbon No. 7).

FIG. 2 is a graph showing geniposide content based on a color value of100 relative to activated carbon treatment time (Test Example 3,Activated carbon No. 8).

FIG. 3 is a graph showing geniposide content based on a color value of100 relative to activated carbon treatment time (Production Test Example1).

FIG. 4 is a graph showing a geniposide content based on a color value of100 relative to activated carbon treatment time (Production Test Example2).

DESCRIPTION OF EMBODIMENTS Terms

In this specification, “color value” means “E_(1cm) ^(10%),” unlessotherwise specified. Further, in this specification, the “color value”is determined according to the method disclosed in Japan'sSpecifications and Standards for Food Additives, 8th Edition (JapanMinistry of Health, Labour and Welfare), unless otherwise specified.

In this specification, “based on a color value of 100” means conversionof a value, such as a measurement value, into a value per 100 colorvalue of the target material, colorant, or the like. For example, whenthe content of geniposide or genipin or both in a material having acolor value of 200 is 500 ppm, the content of geniposide or genipin orboth based on a color value of 100 is determined to be 250 ppm, which isfound by multiplying 500 ppm by the color value ratio (100/200).Although it may be obvious, for example, a measurement value of a samplehaving a color value of 100 can be directly regarded as a value “basedon a color value of 100.”

In this specification, unless otherwise specified, the “roomtemperature” or “normal temperature” means a temperature within a rangeof 10 to 40° C.

In this specification, a “colorant component” means a coloringprinciple.

In this specification, the “colorant” contains a colorant component orconsists only of a colorant component, or may be a mixture of aplurality of colorant components (compounds); further, the “colorant”may contain a raw material-derived component other than a colorantcomponent, a component attributable to the production method, or both.In this specification, the “colorant” is preferably a colorant derivedfrom a natural product. Further, in this specification, the “colorant”can mean an unpurified or roughly purified colorant (or acolorant-containing material), or a purified colorant. This can bejudged by the context.

“Gardenia blue” is defined as follows in Japan's Specifications andStandards for Food Additives, 8th Edition (Japan Ministry of Health,Labour and Welfare). In this specification, the “gardenia blue” may bethose in accordance with this definition.

Definition: “this product is obtained by adding β-glucosidase to amixture of an iridoid glucoside obtained from the fruit of gardenia(Gardenia augusta Merrill or Gardenia jasminoides Ellis) and aproteolytic product, and may contain dextrin or lactose.”

“Gardenia red” is defined as follows in Japan's Specifications andStandards for Food Additives, 8th Edition (Japan Ministry of Health,Labour and Welfare). In this specification, the “gardenia red” may bethose in accordance with this definition.

Definition: “this product is obtained by adding β-glucosidase to amixture of an ester hydrolysate of an iridoid glucoside obtained fromthe fruit of gardenia (Gardenia augusta Merrill or Gardenia jasminoidesEllis) and a proteolytic product, and may contain dextrin or lactose.”

“Gardenia yellow” is defined as follows in Japan's Specifications andStandards of Food Additives, 8th Edition (Japan Ministry of Health,Labour and Welfare). In this specification, the “gardenia yellow” may bethose in accordance with this definition.

Definition: “this product contains, as major components, crocin andcrocetin obtained from the fruit of gardenia (Gardenia augusta Merrillor Gardenia jasminoides Ellis), and may contain dextrin or lactose.”Method for Removing Geniposide or Genipin or Both from MaterialContaining Geniposide or Genipin or Both

The method for removing geniposide or genipin or both from a materialcontaining geniposide or genipin or both of the present invention(hereinafter may also be referred to as a removal method of the presentinvention) is a method comprising treating a material containinggeniposide or genipin or both with an activated carbon having (a) amethylene blue adsorption ability of 50 ml/g or more, and (b) an iodineadsorption ability of 750 mg/g or more, thereby removing geniposide orgenipin or both from the material.

In the present invention, “removal” means complete removal or partialremoval. More specifically, although incomplete removal of geniposide orgenipin or both is regarded as one of the embodiments of the presentinvention, it is preferable to remove geniposide or genipin or bothcompletely or almost completely.

Materials

The amount of the “geniposide or genipin or both” in the “materialcontaining geniposide or genipin or both” to be subjected to the removalmethod of the present invention is not particularly limited, and may beany desired amount to be removed.

The lower limit of the amount is, for example, as the total content ofgeniposide and genipin, 0.02 w/w %, 0.03 w/w %, or 0.04 w/w %, based onthe total solids content of the material.

The upper limit of the amount is, for example, as the total content ofgeniposide and genipin, 2 w/w %, 1.2 w/w %, or 0.6 w/w %, based on thetotal solids content of the material.

The amount is, for example, as the total content of geniposide andgenipin, in a range of 0.02 to 2 w/w %, in a range of 0.03 to 1.2 w/w %,or in a range of 0.04 to 0.6 w/w %, based on the total solids content ofthe material.

When the material is a colorant, the lower limit of the amount is, forexample, as the total content of geniposide and genipin, 350 ppm, 500ppm, or 700 ppm, based on a color value of 100.

When the material is a colorant, the upper limit of the amount is, forexample, as the total content of geniposide and genipin, 5000 ppm, 3000ppm, or 2000 ppm, based on a color value of 100.

When the material is a colorant, the amount is, for example, as thetotal content of geniposide and genipin, in a range of 350 to 5000 ppm,in a range of 500 to 3000 ppm, or in the range of 700 to 2000 ppm, basedon a color value of 100.

In this specification, the amounts of geniposide and genipin aredetermined by HPLC analysis under the conditions specified below, or ananalytical method capable of obtaining results equivalent to the resultsof HPLC.

Conditions of HPLC Analysis

System: JASCO HPLC system

Column: Symmetry C18 (4.6 mm I.D.×250 mm, Waters)

Mobile phase: a) water, b) acetonitrile (b. 12% in 8 min., 12-15% in 2min., 15% in 10 min., 15-100% in 5 min., 100% in 10 min.)Flow rate: 1.0 mL/min.

Temperature: 40° C.

Detection: UV-Vis detector 238 nm

Injection Amount: 20 μL

In the analysis under these analysis conditions, the detection limit ofgeniposide is about 1 ppm, and the detection limit of genipin is alsoabout 1 ppm.

The “material containing geniposide or genipin or both” to be subjectedto the removal method of the present invention is not particularlylimited insofar as the material is “a material containing geniposide orgenipin or both”; however, preferable examples include gardenia-derivedcolorants such as gardenia blue, gardenia red, or gardenia yellow, and agenipap juice or extract.

The “gardenia blue” to be subjected to the removal method of the presentinvention is not particularly limited insofar as it is gardenia bluecontaining geniposide or genipin or both. Examples include gardenia blueproduced by a known production method and gardenia blue that can becommercially obtained.

As described above, “gardenia blue” is generally obtained by subjectingan iridoid glucoside (whose major component is geniposide) obtained byextraction from gardenia fruit of the family Rubiaceae (Gardenia augustaMerrill, or Gardenia jasminoides Ellis) (this may simply be referred toas “gardenia” in this specification) to a β-glucosidase treatment in thepresence of a proteolytic product. In this step, geniposide is convertedinto genipin (aglycone) by the β-glucosidase treatment, and thegenerated genipin is reacted with a proteolytic product, therebygenerating a gardenia blue colorant component.

Therefore, generally, “gardenia blue” is not a single compound, but amixture. “Gardenia blue” generally contains raw material-derivedunreacted geniposide or genipin or both.

The amount of the “geniposide or genipin or both” contained in “gardeniablue” to be subjected to the removal method of the present invention isnot particularly limited; however, the amount is, as the total contentof geniposide and genipin, preferably in a range of 350 to 5000 ppm,more preferably in a range of 500 to 3000 ppm, further preferably in arange of 700 to 2000 ppm, based on a color value of 100. The color valueof gardenia blue is determined by a color value measurement method usinga citrate buffer solution (ph=7.0) as a measurement solvent and amaximum absorption wavelength of 570 to 610 nm.

The “gardenia red” to be subjected to the removal method of the presentinvention is not particularly limited insofar as it is gardenia redcontaining geniposide. Examples include gardenia red produced by a knownproduction method and gardenia red that can be commercially obtained.

As described above, the “gardenia red” is generally obtained bysubjecting an ester hydrolysate (whose major component is geniposidicacid, which is a hydrolysate of geniposide) of an iridoid glucoside(whose major component is geniposide) obtained by extraction fromgardenia fruits of the family Rubiaceae to a β-glucosidase treatment inthe presence of a proteolytic product.

Therefore, generally, “gardenia red” is not a single compound, but amixture. “Gardenia red” generally contains raw material-derivedunreacted geniposide, genipin generated from the unreacted geniposide,or both.

The amount of “geniposide or genipin or both” contained in “gardeniared” to be subjected to the removal method of the present invention isnot particularly limited; however, the amount is, as the total contentof geniposide and genipin, preferably in a range of 350 to 5000 ppm,more preferably in a range of 500 to 3000 ppm, further preferably in arange of 700 to 2000 ppm based on a color value of 100. The color valueof gardenia red is determined by a color value measurement method usingan acetate buffer solution (pH=4.0) as a measurement solvent and amaximum absorption wavelength of 520 to 545 nm.

As described above, gardenia yellow is generally obtained by aqueousextraction or hydrous ethanol extraction of gardenia fruits of thefamily Rubiaceae.

Therefore, gardenia yellow also generally contains raw material-derivedgeniposide.

The amount of “geniposide” contained in “gardenia yellow” to besubjected to the removal method of the present invention is notparticularly limited; however, the amount is, as the total content ofgeniposide and genipin, preferably in a range of 350 to 5000 ppm, morepreferably in a range of 500 to 3000 ppm, further preferably in a rangeof 700 to 2000 ppm, based on a color value of 100. For gardenia yellow,“the total content of geniposide and genipin” is generally equal to thegeniposide content. The color value of gardenia yellow is determined bythe following color value measurement method.

Gardenia Yellow Color Value Measurement Method

About 5 g of a sample based on a color value of 100 is preciselyweighed, and 50 ml of a 0.02 mol/L sodium hydroxide solution is added tothe sample. The mixture is heated for 20 minutes in a 50° C. water bath.As necessary, the mixture is shaken for dissolution, and water is addedto make the amount precisely 100 ml. 1 ml of the resulting solution isprecisely weighed and 50 vol % ethanol is added to make the amountprecisely 100 ml, followed by, as necessary, centrifugation. Theresulting supernatant is used as the test liquid. Absorbency A ismeasured at a maximum absorption wavelength of 410 to 425 nm and at aliquid layer length of 1 cm, and the color value is determined accordingto the formula below. A 50 vol; ethanol is used as a control.

Color value=(A×1000)/collection amount (g)

The amount of “genipin” contained in the genipap (Genipa americana L.)juice (also known as huito juice) or extract to be subjected to theremoval method of the present invention is not particularly limited. Forexample, the amount may be the amount of “genipin” contained in thegeneral huito juice or a genipap extract. The amount as the totalcontent of geniposide and genipin is preferably in a range of 0.02 to0.20 w/w %, more preferably in a range of 0.03 to 0.12 w/w %, furtherpreferably in a range of 0.04 to 0.08 w/w %. For genipap juice orgenipap extract, “the total content of geniposide and genipin” isgenerally equal to the genipin content.

Treatment Using Activated Carbon

The activated carbon used in the removal method of the present inventionis an activated carbon having (a) a methylene blue adsorption ability of50 ml/g or more, and (b) an iodine adsorption ability of 750 mg/g ormore.

The methylene blue adsorption ability of the activated carbon used inthe removal method of the present invention is 50 ml/g or more,preferably 80 ml/g or more, more preferably 100 ml/g or more, furtherpreferably 120 ml/g or more, further more preferably 150 ml/g or more.

The upper limit of the methylene blue adsorption ability of theactivated carbon used in the removal method of the present invention isnot particularly limited; however, the methylene blue adsorption abilityis, for example, generally 1000 ml/g or less, 700 ml/g or less, or 500ml/g or less.

The iodine adsorption ability of the activated carbon used in theremoval method of the present invention is 750 mg/g or more, preferably850 mg/g or more, more preferably 1000 mg/g or more.

The upper limit of the iodine adsorption ability of the activated carbonused in the removal method of the present invention is not particularlylimited; however, the iodine adsorption ability is, for example,generally 4000 mg/g or less, 3000 mg/g or less, or 2500 mg/g or less.

With these values of methylene blue adsorption ability and iodineadsorption ability, geniposide or genipin or both may be highly removed.

The methylene blue adsorption ability of the activated carbon used inthe removal method of the present invention is determined by the methodaccording to the “methylene blue adsorption ability” of JIS K 1474:2014.

The iodine adsorption ability of the activated carbon used in theremoval method of the present invention is determined by the methodaccording to the “iodine adsorption ability” of JIS K 1474:2014.

The upper limit of the “pore volume” of the activated carbon used in theremoval method of the present invention is preferably 1.4 ml/g, morepreferably 1.3 ml/g, further preferably 1.2 ml/g, further morepreferably 1.1 ml/g, particularly preferably 1 ml/g.

With such a range of pore volume, it is possible to selectively remove“geniposide or genipin or both,” while preventing the loss of the usefulcomponents, such as the colorant component of gardenia blue.

The lower limit of the pore volume of the activated carbon used in theremoval method of the present invention is preferably 0.25 ml/g, morepreferably 0.3 ml/g, further preferably 0.5 ml/g, further morepreferably 0.6 ml/g.

With such a range of pore volume, it is possible to highly remove“geniposide or genipin or both.”

The pore volume of the activated carbon used in the removal method ofthe present invention is preferably in a range of 0.25 to 1.4 ml/g, morepreferably in a range of 0.3 to 1.3 ml/g, further preferably in a rangeof 0.5 to 1.2 ml/g, further more preferably in a range of 0.6 to 1.1ml/g, particularly preferably in a range of 0.6 to 1 ml/g.

The pore volume of the activated carbon used in the removal method ofthe present invention is determined as follows.

The Method for Determining Pore Volume

Nitrogen gas is caused to adsorb to a sample (activated carbon) at aliquid nitrogen boiling point, i.e., −195.8° C.

The nitrogen adsorption amount at an equilibrium pressure of P/P₀=0.931is determined and converted into a liquid nitrogen volume, therebydetermining a pore volume.

The lower limit of the “specific surface area” of the activated carbonused in the removal method of the present invention is preferably 650m²/g, more preferably 700 m²/g, further preferably 900 m²/g, furthermore preferably 1000 m²/g.

With such a range of specific surface area, it is possible toselectively remove “geniposide or genipin or both,” while preventing theloss of the useful components, such as the colorant component ofgardenia blue.

The upper limit of the specific surface area of the activated carbonused in the removal method of the present invention is not particularlylimited; however, the specific surface area is generally about 2000m²/g, preferably 1900 m⁴/g, more preferably 1800 m²/g, furtherpreferably 1700 m²/g.

The specific surface area of the activated carbon used in the removalmethod of the present invention is preferably in a range of 650 to 2000m²/g, more preferably in a range of 700 to 1900 m²/g, further preferablyin a range of 900 to 1800 m²/g, further more preferably in a range of1000 to 1700 m²/g.

The specific surface area of the activated carbon used in the removalmethod of the present invention is determined as follows.

Method for Determining Specific Surface Area

Nitrogen gas is caused to adsorb to a sample (activated carbon) at aliquid nitrogen boiling point, i.e., −195.8° C.

The relationship between the pressure and the adsorption amount isdetermined under a relative pressure of 0.1 or less, and a specificsurface area is determined based on the BET theory.

Since such an activated carbon is commercially available, it is possibleto measure the commercially obtained activated carbons using themeasurement methods described above, thereby selecting an activatedcarbon satisfying the above-specified parameters.

Examples of commercially available activated carbons include Kuraraycoal (product name) series from Kuraray Chemical Co., Ltd.; SP seriesfrom Union Service Create Co.; Shirasagi series from JapanEnviroChemicals, Limited; Umebachi jirusi series from Taihei ChemicalIndustrial Co., Ltd.; and activated carbon series from Ueda EnvironmentSolutions Co., Ltd.

The treatment using activated carbon is performed by bringing a materialcontaining geniposide or genipin or both into contact with activatedcarbon. As a result, geniposide or genipin or both adsorb to theactivated carbon. Further, by removing the activated carbon in whichgeniposide or genipin or both is adsorbed, it is possible to removegeniposide or genipin or both.

When the material containing geniposide or genipin or both is a solid,the contact of the material with activated carbon may be performed, forexample, by dissolving or suspending the material in an aqueous solventto prepare a liquid-form material, and mixing the liquid material withactivated carbon. When the material containing geniposide or genipin orboth is a liquid, the contact of the material with activated carbon maybe performed, for example, by directly mixing the liquid material withactivated carbon, or first diluting the liquid material with an aqueoussolvent and then mixing it with activated carbon. The mixing may beperformed by a conventional method using a shaking apparatus, a stirrer,and the like.

Examples of the aqueous solvents include water (e.g., tap water,ion-exchanged water, distilled water) and hydrous alcohol (e.g., hydrousethanol). In terms of preventing adsorption of useful components, suchas the gardenia blue colorant component, to activated carbon, thealcohol (e.g., ethanol) content in the aqueous solvent is preferablysmall, and the aqueous solvent is preferably water (e.g., tap water,ion-exchanged water, distilled water).

The pH of the liquid material is not particularly limited; however, thepH is generally in a range of 2.0 to 7.0, preferably in a range of 4.0to 7.0.

When pH of the liquid material is adjusted, the adjustment may beperformed by a conventional method using hydrochloric acid or sodiumhydroxide.

The temperature in the treatment using activated carbon is notparticularly limited; however, the treatment is generally performed atroom temperature.

The appropriate amount of the activated carbon used for the activatedcarbon treatment varies depending on the type of material containinggeniposide or genipin or both, the types and the contents of usefulcomponents in the material, the amount of geniposide and genipin or bothin the material, and the like. The amount of the activated carbon in aliquid material is, for example, in a range of 0.1 to 10%, morepreferably 1 to 7%. For gardenia blue having a total content ofgeniposide and genipin in a range of 350 to 5000 ppm per 100 colorvalue, the amount of the activated carbon in the liquid material ispreferably in a range of 1 to 10 w/w %, more preferably in a range of 2to 8 w/w %, further preferably in a range of 3 to 7 w/w %.

The duration of the activated carbon treatment is not particularlylimited; however, the duration is generally 10 minutes to 50 hours,preferably 30 minutes to 40 hours, more preferably in a range of 1 to 30hours. During this time period, the above mixing state is preferablymaintained.

If the duration is excessively short, the removal of “geniposide andgenipin or both” will be insufficient; on the other hand, if theduration is excessively long, the removal ratio of “geniposide andgenipin or both” will not increase, which is disadvantageous in terms ofwork efficiency.

Membrane Treatment

In the removal method of the present invention, the material containinggeniposide or genipin or both is preferably first treated with amembrane before the activated carbon treatment is performed.

The “geniposide or genipin or both” can be removed to some extent by themembrane treatment. Thus, by conducting the membrane treatment, forexample, it is possible to reduce the amount of activated carbonrequired in the activated carbon treatment.

The membrane used for the membrane treatment is preferably anultrafilter membrane; more preferably, the membrane has a molecularweight cut-off in a range of 2000 to 5000. Such a membrane may becommercially obtained. The membrane treatment using an ultrafiltermembrane is preferably performed, for example, for 3 to 24 hours at 0.3to 0.7 MPa.

When the membrane treatment is performed, the “material containinggeniposide or genipin or both” to be subjected to the activated carbontreatment is a concentrated liquid obtained by the membrane treatment.The concentrated liquid may be subjected to other treatments, such asdilution or concentration, as necessary, before being subjected to theactivated carbon treatment.

The removal method of the present invention makes it possible to reducethe amount of geniposide or genipin or both in the material whilepreventing the loss of the useful components such as gardenia blue inthe material.

In the removal method of the present invention, the residual ratio ofgeniposide and genipin in terms of the total content (i.e., the massratio of the total content after the treatment of the removal method ofthe present invention with respect to the total content before thetreatment) is preferably 65% or less, more preferably 20% or less,further preferably 5% or less, further more preferably 1% or less,particularly preferably 0.5% or less, most preferably 0%.

In a preferred embodiment of the present invention in which “thematerial containing geniposide or genipin or both” is a gardenia-derivedcolorant, the residual ratio of the colorant component, i.e., a usefulcomponent (i.e., the ratio of the content of the colorant componentafter the treatment of the removal method of the present invention withrespect to the content before the treatment), is preferably 85% or more,more preferably 90% or more, further preferably 95% or more, furthermore preferably 98% or more; and the residual ratio of geniposide andgenipin in terms of the total content is preferably 65% or less, morepreferably 20% or less, further preferably 5% or less, further morepreferably 1% or less, particularly preferably 0.5% or less, mostpreferably 0% (including the case in which the content of geniposide andgenipin in the gardenia-derived colorant after the treatment of theremoval method of the present invention is less than the detectionlimit, and the residual ratio is thus regarded as 0%).

Examples of colorant components include gardenia blue components,gardenia red components, and gardenia yellow components.

In a particularly preferred embodiment of the present invention, thecolorant component is a gardenia blue component.

The content of the colorant component is determined based on the colorvalue. Therefore, “a residual ratio of colorant component” may beregarded as “a residual ratio of color value.”

The “residual ratio of color value” is defined as follows.

Residual ratio of color value (%)=(color value after the treatment ofthe removal method of the present invention)/(color value before thetreatment of the removal method of the present invention)×100

Method for Producing Gardenia-Derived Colorant in Which Total Content ofGeniposide and Genipin is Reduced

The removal method of the present invention described above removesgeniposide or genipin or both from a gardenia-derived colorantcontaining geniposide or genipin or both, thereby producing agardenia-derived colorant in which the total content of geniposide andgenipin is reduced. More specifically, an embodiment of the presentinvention is a method for producing a gardenia-derived colorant in whichthe total content of geniposide and genipin is reduced.

Examples of the gardenia-derived colorants include gardenia blue,gardenia red, and gardenia yellow.

The expression “reduced” means reduction in the total content ofgeniposide and genipin in the material, and may mean that the materialis free of, or substantially free of, geniposide and genipin.

In the production method of the present invention, the solution obtainedafter the treatment with activated carbon may be further subjected to aconventional purification treatment such as, filtration, resintreatment, or membrane treatment.

Gardenia-Derived Colorant, Gardenia-Derived Colorant Preparation,Gardenia-Derived Colorant Color Composition

The gardenia-derived colorant obtained by the production method of thepresent invention may be in the form of a solution obtained after theactivated carbon treatment, a concentrate thereof, or in the form ofpowder obtained by drying the solution in an arbitrary method (e.g.,vacuum drying, freeze-drying, spray-drying, or the like).

The amount of the “geniposide or genipin or both” in thegardenia-derived colorant obtained by the production method of thepresent invention is, as the total content of geniposide and genipin,preferably 300 ppm or less, more preferably 200 ppm or less, furtherpreferably 100 ppm or less, further more preferably 50 ppm or less,particularly preferably 20 ppm or less, more particularly preferably 10ppm or less, further particularly preferably 5 ppm or less, further moreparticularly preferably 1 ppm or less, based on a color value of 100.

The amount of the “geniposide or genipin or both” is preferablymaximally reduced. In particular, the amount is preferably reduced to beless than the measurement limit in the measurement method describedabove. Therefore, the lower limit of the amount is not limited. However,for example, an amount of 1 ppm or more, 2 ppm or more, or 5 ppm or morein the gardenia-derived colorant is acceptable depending on the purposeand the form of use.

An embodiment of the present invention is gardenia blue having a totalcontent of geniposide and genipin based on a color value of 100 of 300ppm or less (more preferably 200 ppm, further preferably 100 ppm orless, further more preferably 50 ppm or less, particularly preferably 10ppm or less, further particularly preferably 5 ppm or less, more furtherparticularly preferably 1 ppm or less, most preferably 0 ppm (or lessthan the detection limit in the measurement method described above)).The gardenia blue may be obtained by the production method of thepresent invention described above.

The gardenia-derived colorant obtained by the production method of thepresent invention and the gardenia blue of the present invention may beused in a manner similar to that of the previously knowngardenia-derived colorants, and may be provided as a colorantpreparation either as is or after being mixed with other components suchas a diluent, a carrier, or other additives.

Examples of diluents, carriers, and additives include various generalcomponents used for a colorant preparation, in particular, awater-soluble colorant preparation, insofar as the advantageous effectsof the prevent invention are not impaired.

Examples include sucrose, lactose, glucose, dextrin, gum arabic, water,ethanol, propylene glycol, glycerin, and starch syrup.

The colorant preparation may be prepared into an arbitrary form, suchas, but not particularly limited to, powder, granules, tablet, liquid,emulsion, or paste.

The colorant preparation of the present invention may be widely used ascolorants for food, perfumery and cosmetics, pharmaceuticals,quasi-drugs, feedstuff, and the like in a manner similar to that of thepreviously known gardenia-derived colorant preparations.

The present invention provides a color composition, such as food,perfumery and cosmetics, pharmaceuticals, quasi-drugs, or feedstuff,that is colored by using the gardenia-derived colorant described aboveor the colorant preparation thereof.

Examples of food include confectioneries such as frozen dessert, freshcakes, Japanese confectioneries, or western confectioneries; beveragessuch as beverages and alcohol beverages; farm-processed products such asdehydrated vegetables or pickles; processed seafood; and processed meat.

Examples of the perfumery and cosmetics include cosmetics (e.g., eyeshadow, mascara, lipstick, lip balm, lotion, or the like), soap,shampoo, conditioner, detergent, toothpaste, and mouthwash.

Examples of pharmaceuticals include tablets (e.g., sugar-coatedtablets), granules, liquids, and capsules.

The content of the gardenia-derived colorant in these color compositionsis not particularly limited; however, for example, the content ofgardenia blue is generally set so that the absorbency of the colorcomposition is 0.01 to at the maximum absorption wavelength, which isabout 605 nm.

Method for Producing Genipap Juice or Genipap Extract in which GenipinContent is Reduced

The removal method of the present invention described above is capableof removing genipin from a genipap juice or extract containing genipin(as stated above, a genipap juice or extract generally containsgenipin), thereby producing a genipap juice or extract in which thegenipin content is reduced. More specifically, an embodiment of thepresent invention is a method for producing a genipap juice or extractin which the genipin content is reduced.

The expression “reduced” means reduction in the total content ofgeniposide and genipin in the material, and may mean that the materialis free of, or substantially free of geniposide and genipin.

The “genipap juice or extract in which the genipin content is reduced”obtained by the “method for removing geniposide or genipin or both froma material containing geniposide or genipin or both” described above maybe used in a manner similar to that of the previously known genipapjuice or extract either as is or after being subjected to purifications,processing, and the like as desired.

EXAMPLES

The present invention is described in more detail below with referenceto Examples. However, the present invention is not limited to theExamples.

Each symbol and abbreviation stands for the following. Aside from thesesymbols and abbreviations, symbols and abbreviations generally used inthe related technical field are used in this specification.

CV: Color Value

Regarding “the total content of geniposide and genipin” or the like inthe description below, “geniposide and genipin” may be referred to as“Total G.”

For example, “Total G residual ratio” is defined as follows.

Total G residual ratio (%)=(geniposide amount after treatment+genipinamount after treatment)/(geniposide amount before treatment+genipinamount before treatment)×100

In the Test Examples and the Examples below, quantitative determinationsof geniposide and genipin were performed by HPLC analysis under thefollowing conditions.

When an analysis sample having a color value over 100 was used in thisanalysis, the analysis sample was diluted with ultrapure water so as toadjust the color value to 100.

Conditions of HPLC Analysis

System: JASCO HPLC System Column: Symmetry C18 (4.6 mm I.D.×250 mm,Waters)

Mobile phase: a) water, b) acetonitrile (b. 12% in 8 min., 12-15% in 2min., 15% in 10 min., 15-100% in 5 min., 100% in 10 min.)Flow rate: 1.0 mL/min.

Temperature: 40° C.

Detection: UV-Vis detector 238 nmInjection amount: 20 μL

In the analysis under the above conditions, if geniposide or genipin wasnot detected (i.e., if the value was less than the detection limit), thegeniposide concentration or the genipin concentration in the calculationof the total content of geniposide and genipin were regarded as 0 ppm.

Test Example 1 Geniposide Adsorption Test Using Various ActivatedCarbons

Geniposide Adsorption Tests were Performed Using various activatedcarbons (activated carbon Nos. 1 to 16). These activated carbons wereobtained from commercial suppliers. Activated carbons No. 5 and No. 6were the same product, with different lot numbers.

Each activated carbon was added to a solution of about 500 ppmgeniposide at a ratio of 1 w/w %. After 1-hour shaking, the geniposidecontent was measured under the HPLC analysis conditions detailed above.

Table 1 shows the results. In Table 1, the “control” was not treatedwith activated carbon, and “ND” means less than the detection limit.

As shown in Table 1, the geniposide adsorption ability significantlyvaried depending on the type of activated carbon.

TABLE 1 Activated Geniposide Geniposide Carbon Concentration ResidualRatio No. (ppm) (%) Control 509.2 100.0 1 2.2 0.4 2 2.2 0.4 3 6.4 1.3 44.6 0.9 5 2.3 0.5 6 2.3 0.4 7 2.6 0.5 8 463.7 91.1 9 ND 0 10 362.6 71.211 ND 0 12 479.4 94.1 13 2.8 0.5 14 2.1 0.4 15 11.3 2.2 16 2.4 0.5

Test Example 2 Geniposide Adsorption Test in the Presence of GardeniaBlue

Nine kinds of activated carbons (activated carbon Nos. 4, 6, 7, 9, 11,13, 14, 15, and 16) were selected from the activated carbons that wereconfirmed to have high geniposide adsorption ability in Test Example 1,and a geniposide and genipin adsorption test was performed using theseactivated carbons in the presence of gardenia blue for the considerationof their actual ability to remove geniposide and genipin from gardeniablue. Since genipin was not detected from the gardenia blue used in thistest, this test actually confirmed the geniposide adsorption toactivated carbon. However, since genipin is similar to geniposide in itsstructure and physicochemical property, genipin is reasonably assumed tobehave similarly to geniposide in the Test Examples and the Examples.

β-glucosidase was caused to act on purified geniposide (geniposidecontent=91705 ppm) derived from a gardenia fruit in the presence of aprotein hydrolysate, thereby obtaining gardenia blue (geniposidecontent=1372 ppm, genipin content=less than the detection limit).Purified geniposide (geniposide content=34.8 w/w %, genipin content=lessthan the detection limit) derived from a gardenia fruit was added to theresulting gardenia blue so that the geniposide content was about 2000ppm, thereby preparing geniposide-containing gardenia blue. Eachactivated carbon was added the resulting geniposide-containing gardeniablue (specifically, 0.2 g of activated carbon was added to 20 g ofgeniposide-containing gardenia blue (this colorant was made into asolution adjusted with water to have a color value of 100)) at a ratioof 1 w/w %, and the mixture was shaken at normal temperature. After anhour, the color value and the geniposide content were measured.

Table 2 shows the results. In Table 2, the “control” was not treatedwith activated carbon.

As shown in Table 2, the geniposide adsorption ability and the colorvalue adsorption ability varied depending on the type of activatedcarbon, and the geniposide content calculated based on a color value of100 (geniposide/CV100) also varied. However, some activated carbonsselectively adsorbed geniposide in the gardenia blue. It was thussuggested that these activated carbons may ensure a high color valueresidual ratio and high geniposide removal at the same time.

TABLE 2 Color Geniposide Total G Value Activated Geniposide GenipinResidual Residual Residual Geniposide/ Total G/ Carbon ConcentrationConcentration Ratio Ratio Color Ratio CV100 CV100 No. (ppm) (ppm) (%)(%) Value (%) (ppm) (ppm) Control 2047.9 ND 100.0 100.0 101.2 100.0 20242024 4 1438.0 ND 70.2 70.2 97.8 96.6 1470 1470 6 1643.1 ND 80.2 80.296.4 95.3 1704 1704 7 1283.6 ND 62.7 62.7 100.6 99.4 1276 1276 9 1131.4ND 55.2 55.2 101.0 99.8 1120 1120 11 1728.1 ND 84.4 84.4 102.3 101.11689 1689 13 1858.8 ND 90.8 90.8 101.9 100.7 1824 1824 14 1658.2 ND 81.081.0 101.6 100.4 1632 1632 15 1883.5 ND 92.0 92.0 98.5 97.3 1912 1912 161810.5 ND 88.4 88.4 101.2 100.0 1789 1789

Test Example 3 Consideration Regarding Amount of Activated Carbon andActivated Carbon Treatment Time

Consideration regarding the amount of activated carbon and the activatedcarbon treatment time was performed.

Activated carbons No. 7 and No. 8 were used for the consideration.Activated carbon No. 7 was confirmed for a low geniposide content basedon a color value of 100 (geniposide/CV100) in Test Example 2. Activatedcarbon No. 8 was confirmed for a high geniposide residual ratio in TestExample 1.

A test similar to that of Test Example 2 was performed, except that,after activated carbon No. 7 or No. 8 was added at a ratio of 1 w/w %, 3w/w % or 5 w/w %, the mixture was stirred at normal temperature with astirrer, and the color value and the geniposide content were measuredafter an hour, after 3 hours and after 5.5 hours. With these measurementvalues, geniposide contents based on a color value of 100(geniposide/CV100) were calculated. FIG. 1 (activated carbon No. 7) andFIG. 2 (activated carbon No. 8) show the results.

Further, the geniposide adsorption amount (mg) of activated carbon No. 7was measured. With the measurement value, the geniposide adsorptionamount per unit mass of the activated carbon (geniposide adsorptionamount (g)/per gram of activated carbon) was calculated. Table 3 showsthe results.

TABLE 3 Geniposide Geniposide Adsorption Activated Adsorption Amount(g)/1 g carbon Amount of Activated Amount (mg) Carbon 1 w/w % 19.300.096 3 w/w % 38.20 0.064 5 w/w % 39.91 0.040

As shown in FIG. 1, when activated carbon No. 7 was used, after 5.5hours, geniposide was removed to about 1000 ppm/CV100 by 1 w/w %addition, and to about 30 ppm/CV100 by 5 w/w % addition.

The maximum geniposide adsorption amount of activated carbon No. 7 isassumed to be about 0.1 g/g. The results of Table 3 revealed that it ispreferable to add an excessive amount of activated carbon to ensureefficient removal. Regarding the treatment time, there was a tendencythat the decrease in geniposide content based on a color value of 100(geniposide/CV100) became moderate when the treatment time exceeded anhour.

In contrast, as shown in FIG. 2, when activated carbon No. 8 was used,the geniposide content based on a color value of 100 (geniposide/CV100)was not decreased by increasing the addition amount or extending thetreatment time. The results revealed that the geniposide adsorptionamount was not increased simply by increasing the addition amount ofactivated carbon and/or extending the activated carbon treatment time,and that selection of activated carbon based on appropriate factors wasimportant.

Production Test Example 1 Production of Geniposide-Reduced Gardenia Blue1 Step 1

β-glucosidase was caused to act on purified geniposide (geniposidecontent=37.5 w/w %) derived from a gardenia fruit in tap water in thepresence of a protein hydrolysate, thereby preparing gardenia blue(solution state, liquid measure=2350 g, color value=113.3, geniposidecontent based on a color value of 100=1372 ppm, genipin content=lessthan the detection limit).

The obtained gardenia colorant was filtrated under the followingfiltration conditions, thereby obtaining gardenia blue as the filtrate(solution state, liquid measure=2606 g, color value=100.9).

Filtration Conditions

Filter Paper: ADVANTEC No. 2, 4125 mm (ADVANTEC)

Filtration adjuvant: diatomaceous earthFiltration adjuvant precoating amount: 20 gFiltration adjuvant body-feed amount: 2 w/w % of liquid measure

Step 2

The gardenia blue after the filtration using the above filter paper wastreated with a ultrafilter membrane (0.5 MPa, 15 hours and 40 minutes),thereby obtaining gardenia blue (solution state, liquid measure=2448 g,color value=102.6, solids content=14.6%, color value/solidscontent=704.2, yield=95.5%, geniposide content based on a color value of100=432 ppm, genipin content=less than the detection limit).

Step 3

Activated carbon No. 9 was added to the gardenia blue after theultrafiltration at a ratio of 1 w/w %, 3 w/w % or 5 w/w %, and themixture was stirred at normal temperature with a stirrer, and the colorvalue and the geniposide content were measured after an hour, after 3hours, and after 5 hours. Table 4 shows colorant residual ratios (after5 hours) calculated from these measurement values. Further, Table 4 andFIG. 3 show graphs of the total content of geniposide and genipin (TotalG/CV100) based on a color value of 100 calculated from these measurementvalues.

TABLE 4 Total Colorant Activated G/CV100 Total Total Total ResidualCarbon (Before G/CV100 G/CV100 G/CV100 Ratio Amount Treatment) (1 h) (3h) (5 h) (5 h) 1 w/w % 432 ppm 321 ppm 268 ppm 262 ppm 100%  3 w/w % 432ppm 135 ppm  84 ppm  57 ppm 97% 5 w/w % 432 ppm  4 ppm  3 ppm  3 ppm 95%

As shown in Table 4 and FIG. 3, there was a tendency for the totalcontent of geniposide and genipin based on a color value of 100 (TotalG/CV100) to decrease as the amount of the activated carbon increased.Regarding the treatment time, there was a tendency that the decrease inthe total content of geniposide and genipin based on a color value of100 (Total G/CV100) became moderate when the treatment time exceeded anhour.

As is clear from Table 4, there was a tendency for the color valueresidual ratio to decrease as the amount of the activated carbonincreased.

Production Test Example 2

For the consideration of the difference in the adsorption effectdepending on the presence or absence of alcohol, solutions were preparedby adding 20 parts by weight of water or ethanol to 80 parts by weightof gardenia blue obtained after the ultrafiltration in Production TestExample 1, and activated carbon No. 9 was added thereto at a ratio of 1w/w %. The mixture was stirred at normal temperature with a stirrer, andthe color value and the geniposide content were measured after 5 hours.Table 5 shows colorant residual ratios calculated from these measurementvalues.

TABLE 5 Test Fraction Colorant Residual Ratio (5 h) Water added (Ethanolnot added) 98% Ethanol added 93%

Further, FIG. 4 shows a graph of the total content of geniposide andgenipin based on a color value of 100 (Total G/CV100) in the testfraction without ethanol and the test fraction with ethanol in whichactivated carbon was added at a ratio of 1 w/w %.

As shown in FIG. 4, there was a tendency that, by the addition ofethanol, the colorant yield decreased and the total content ofgeniposide and genipin based on a color value of 100 (Total G/CV100)increased.

The results thus revealed that addition of no ethanol or a small amountof ethanol is preferred to achieve the object of the present invention.

Test Example 4 Analysis Regarding Relationship Between Nature ofActivated Carbon, Geniposide Residual Ratio, and Color Value ResidualRatio

The relationship between the nature of activated carbon, geniposideresidual ratio, and color value residual ratio was analyzed.

The shapes, the raw materials, the activation methods, and theproperties of the activated carbons used in Test Examples 1 and 2 wereexamined and analyzed, and the relationship between these natures ofactivated carbons, the geniposide residual ratio, and the color valueresidual ratio was analyzed.

Table 6 shows the results of these examinations and analyses. In Table6, “Control” was not treated with activated carbon, and “ND” means lessthan the detection limit.

TABLE 6 Test Test Example Example 2 Methylene 1 Color Iodine BlueSpecific Geniposide Value Activated Adsorption Adsorption Surface PoreResidual Residual Carbon Raw Activation Ability Ability Area VolumePatio Patio No. Shape Material Method (mg/g) (ml/g) (m²/g) (ml/g) (%)(%) Control — — — — — — — — — 12 Powder Palm Water 710 30 620 0.28 94.1— Shell Vapor 8 Granules Palm Water 710 30 620 0.28 91.1 — Shell Vapor10 Powder Palm Water 710 30 620 0.28 71.2 — Shell Vapor 4 Granules WoodZinc 950 150 1341 1.12 0.9 96.6 Flour Chloride 16 Powder Wood Water 950100 1045 0.61 0.5 100 Flour Vapor 6 Powder Wood Zinc 950 150 1516 1.370.45 95.3 Flour Chloride 2 Powder Wood Zinc 950 150 1496 1.44 0.4 —Flour Chloride 1 Powder Wood Zinc 960 160 1556 1.46 0.4 — Flour Chloride15 Powder Wood Zinc 1000 160 1555 1.49 2.2 97.3 Flour Chloride 14 PowderWood Water 1020 160 1151 0.78 0.4 100.4 Flour Vapor 13 Powder Palm Water1080 120 1031 0.48 0.5 100.7 Shell Vapor 11 Powder Palm Water 1280 1701150 0.51 ND 101.1 Shell Vapor 9 Powder Palm Water 1550 230 1660 0.78 ND99.8 Shell Vapor 3 Granules Palm Water 1570 310 1707 0.72 1.3 — ShellVapor 7 Granules Palm Water 1610 320 1600 0.74 0.5 99.4 Shell Vapor

As shown in Table 6, there was a tendency that activated carbons havinghigh methylene blue adsorption ability and high iodine adsorptionability selectively adsorbed geniposide.

Further, there was also a tendency that activated carbons having a porevolume of less than 1.12 had a significantly high gardenia blue colorantresidual ratio.

Further, the specific surface area is preferably greater to ensuregreater adsorption of geniposide. In particular, the results showed thatthe geniposide residual ratio based on a color value of 100 was low whenthe specific surface area was 650 m²/g or more.

Test Example 5 Gardenia Yellow

0.57 g of purified geniposide (geniposide content=34.8 w/w %) derivedfrom a gardenia fruit was added to 100 g of a 5% solution of crocin(Tokyo Chemical Industry Co., Ltd.) so that the geniposide content wasabout 2000 ppm. 20 g each of the mixture was placed in three 50-ml screwtubes, which were respectively used as a control (not treated withactivated carbon), a fraction with activated carbon No. 7, and afraction with activated carbon No. 8. The addition amount of theactivated carbon was 5 w/w % (1 g each).

Each sample was stirred for three hours at normal temperature, and thecolor value and the geniposide content were measured. Table 7 showscolor value residual ratios and geniposide residual ratios calculatedfrom these measurement values.

TABLE 7 Color Value Residual Geniposide Residual Test Fraction Ratio (%)Ratio (%) Control 96 100 Activated Carbon 87 4 No. 7 Activated Carbon 9390 No. 8

As shown in Table 7, when the useful component was gardenia yellow aswell, as in the case of gardenia blue, geniposide was selectivelyremoved while preventing the loss of the useful component when activatedcarbon No. 7 was used. In contrast, when the useful component wasgardenia yellow as well, as in the case of gardenia blue, the geniposideremoval failed when activated carbon No. 8 was used, although the colorvalue residual ratio was high.

Reference Example 1 Analysis of Commercially Available Gardenia Blue

Four kinds of commercially available gardenia blue were analyzed to becompared with the gardenia blue of the present invention.

Table 8 shows the results. In Table 8, “ND” is less than the detectionlimit.

TABLE 8 Color Geniposide/CV100 Genipin/CV100 Total G/CV100 Product Value(ppm) (ppm) (ppm) A 218.2 330 ND 330 B 189.0 783 ND 783 C 186.2 552 ND532 D 190.9 467 ND 467

As is clearly shown in Table 8, the total content of geniposide andgenipin in a commercially available gardenia blue based on a color valueof 100 was at least more than 300 ppm.

Reference Example 2

Consideration of Geniposide Removal from Gardenia Blue UsingAnion-Exchange Resin

The possibility of geniposide removal from gardenia blue using ananion-exchange resin was considered.

A geniposide removal test was performed using a weakly basicanion-exchange resin IRA96SB (product name, Organo Corporation) using acolumn method. This resin had been confirmed for a certain geniposideadsorption effect in a batch-type preliminary test.

The test was performed as follows.

Resin Conditioning

IRA96SB was immersed in ion-exchanged water overnight to be swollen, and20 ml of the swollen resin was placed in a column (3 columns).

Adsorption Test

Test Liquid A: about 30 ppm solution of geniposide sample (pH=6.18)Test Liquid B: gardenia blue liquid (color value=82.6) (pH=6.04)Test Liquid C: gardenia blue liquid (color value=82.6) (pH=9.30,adjusted with 48% NaOH).

20.00 g each of Test Liquids A to C were fed at a velocity of about 2 SV(space velocity), and water-washing was performed by feedingion-exchanged water in an amount three times (60 ml) the amount of eachresin at a velocity of about 4 SV. All liquids from the beginning of theliquid feed to the end of the water-washing were collected (about 80 g).

The geniposide content of each sample was measured by HPLC.

Table 9 shows the results. In Table 9, the numbers (%) in parenthesesdenote a recovery ratio.

TABLE 9 Total G/ Liquid Geniposide Genipin CV100 Total G Amount ColorConcentration Concentration Concentration Amount (g) Value (ppm) (ppm)(ppm) (μg) pH Before Test Liquid A 20.00 — 58 ND — 1160 6.18 Resin TestLiquid B 20.00 82.55 1486 ND 1800 29720 6.04 Treatment Test Liquid C20.00 82.55 1486 ND 1800 29720 9.30 After Test Liquid A 83.49 — 15 ND —1252 2.96 Resin (108%) Treatment Test Liquid B 82.81 11.18 295 ND 263824429 4.81 (82%) Test Liquid C 81.89 12.44 305 ND 2451 24976 5.00 (84%)

As shown in Table 9, a geniposide removal effect was not observed in anyof the geniposide sample (Test Liquid A) and the gardenia blue liquids(Test Liquids B and C). Rather, in the test using gardenia blue liquids(Test Liquids B and C), the geniposide recovery ratio were about 80%, incontrast to the colorant recovery ratio of about 60%; in addition, thegeniposide concentration based on a color value of 100 increased afterthe resin treatment.

Further, although the adsorption difference depending on the pH in theliquid feed was not clearly observed, assuming, inconveniently, theactual step, there was a tendency that precipitates were more easilygenerated in the resin and the recovered liquid when the pH was low.

Therefore, after this test, it was concluded that geniposide removalfrom gardenia blue using a weakly basic anion-exchange resin isdifficult.

Reference Example 3

Consideration of Geniposide Removal from Gardenia Blue by AcidPrecipitation or Base Precipitation

The possibility of geniposide removal from gardenia blue by acidprecipitation or base precipitation was considered.

The test was performed as follows.

Preparation of Gardenia Blue

β-glucosidase was caused to act on purified geniposide (geniposidecontent=36.9 w/w %) derived from a gardenia fruit in the presence of aprotein hydrolysate, thereby obtaining gardenia blue (solution state,color value=118.1, geniposide content=2288 ppm).

Acid Precipitation

50 g of sterilized gardenia blue was adjusted to have a pH of 2.5 using97% sulfuric acid. The mixture was stirred with a stirrer, and keptstill overnight at normal temperature. The supernatant and theprecipitate were transferred to a centrifuging tube, and subjected to3000G×20 minutes centrifugation. The supernatant was isolated. Theresidual precipitate was washed twice with sulfuric acid water adjustedto pH 2.5, and further washed once with ion-exchanged water. Anappropriate amount of ion-exchanged water was added to the washedprecipitate, thereby dissolving a part of the precipitate. At this time,an appropriate amount of 47% NaOH was added to dissolve the precipitate.

The geniposide contents in the resulting supernatant and the precipitatewere measured by HPLC.

Base Precipitation

23.6 g of ammonium sulfate was added to 50 g of sterilized gardeniablue. The mixture was stirred with a stirrer, and kept still overnightat normal temperature. The supernatant and the precipitate weretransferred to a centrifuging tube, and subjected to 3000G×20 minutescentrifugation. The supernatant was isolated. The residual precipitatewas washed twice with ammonium sulfate water having a saturation degreeof 70%, and further washed once with ion-exchanged water. An appropriateamount of ion-exchanged water was added to the washed precipitate,thereby dissolving a part of the precipitate.

The geniposide contents in the resulting supernatant and the precipitatewere measured by HPLC.

Table 10 shows the results. In Table 10, since the precipitate dilutionratio is not precise, the numbers in parentheses show reference values.

TABLE 10 Acid Precipitation Base precipitation Before Super- Precip-Super- Precip- Treatment natant itate natant itate Color 118.1 27.3   (5.3) 0.9    (72.0) Value Geniposide 2288 2008  (69) 242 (1146)Content (ppm) Geniposide/ 1938 7356 1304 26931 1591 CV100 (ppm)

As is clearly shown in Table 10, the geniposide contents based on acolor value of 100 (geniposide/CV100) were large in both the supernatantand the precipitate in the acid precipitation and the baseprecipitation. It was thus concluded that it is difficult to removegeniposide and genipin from a colorant component by acid precipitationor base precipitation.

Production Test Example 3 Production of Geniposide-Reduced Gardenia Blue2 Step 1

β-glucosidase was caused to act on purified geniposide (geniposidecontent=35.7 w/w %) derived from a gardenia fruit in tap water in thepresence of a protein hydrolysate, thereby preparing gardenia blue(solution state, liquid measure=1200 L, color value=105.2, geniposidecontent based on a color value of 100=597.4 ppm, genipin content=lessthan the detection limit).

The obtained gardenia colorant was filtrated under the followingfiltration conditions, thereby obtaining gardenia blue as the filtrate(solution state, liquid measure=1900 L, color value=65.2).

Filtration Conditions

Filtration facility: filter pressFiltration adjuvant: diatomaceous earthFiltration adjuvant precoating amount: 30 kgFiltration adjuvant body-feed amount: 20 kg

Step 2

The gardenia blue after the filtration in the filtration facility wastreated (0.5 MPa, 5 hours) with an ultrafilter membrane (molecularweight cut-off=3000), thereby obtaining gardenia blue (solution state,liquid measure=1300 L, color value=96.0, solids content=11.7%, colorvalue/solids content=820.5, yield=98.9%).

Step 3

Activated carbon No. 9 was added to the gardenia blue after theultrafiltration in an amount of 3 w/w %, followed by cooling to 15° C.,2-hour stirring with a stirrer, and standing still overnight.

The obtained gardenia blue was filtrated under the following filtrationconditions, thereby obtaining gardenia blue as the filtrate (solutionstate, liquid measure=2800 L, color value=41.7).

Filtration Conditions

Filtration facility: filter pressFiltration adjuvant: diatomaceous earthFiltration adjuvant precoating amount: 30 kgFiltration adjuvant body-feed amount: 58 kg

Step 4

The filtrate obtained through the filtration in step 3 containinggardenia blue was concentrated under reduced pressure, thereby obtaininggardenia blue (color value=242.9, liquid measure=343 kg).

The geniposide and genipin contents of the gardenia blue were measured,and the results showed that the geniposide content was 1.6 ppm (0.7ppm/CV100) and the genipin content was less than the detection limit.

More specifically, the total content of geniposide and genipin based ona color value of 100 (Total G/CV100) was 0.7 ppm/CV100.

Test Example 6 Gardenia Red

β-glucosidase was caused to act on an ester hydrolysate of purifiedgeniposide derived from a gardenia fruit in the presence of a proteinhydrolysate, and purified geniposide derived from a gardenia fruit(geniposide content=36.5 w/w %, genipin content=less than the detectionlimit) was added to the resulting gardenia red so that the geniposidecontent became about 2000 ppm, thereby preparing geniposide-containinggardenia red.

20 g each of the geniposide-containing gardenia red (this colorant wasmade into a solution adjusted with water to have a color value of 100)was placed in three 50-ml screw tubes, which were respectively used as acontrol (not treated with activated carbon), a fraction with activatedcarbon No. 7, and a fraction with activated carbon No. 8. The additionamount of the activated carbon was 5 w/w % (1 g each).

Each sample was stirred for three hours at normal temperature, andfiltrated with a 0.2 μm filter, thereby obtaining a sample.

The color values and the geniposide contents of the filtrated sampleswere measured. Table 11 shows color value residual ratios and geniposideresidual ratios calculated from these measurement values. In Table 11,“ND” means less than the detection limit.

TABLE 11 Color Value Geniposide Residual Geniposide Geniposide ResidualRatio Content Content/CV100 Ratio (%) (ppm) (ppm) (%) Control 101.91913.3 1877.6 100 Activated 103.1 ND ND 0 Carbon No. 7 Activated 105.41827.8 1734.2 92 Carbon No. 8

As shown in Table 11, when the useful component was gardenia red aswell, as in the case of gardenia blue, geniposide was selectivelyremoved while preventing the loss of the useful component when activatedcarbon No. 7 was used. In contrast, as in the case of gardenia blue, thegeniposide removal failed when activated carbon No. 8 was used, althoughthe color value residual ratio was high.

1-7. (canceled)
 8. A colorant preparation comprising a gardenia-derivedcolorant produced by a method comprising treating a material comprisinggeniposide or genipin or both with an activated carbon having: (a) amethylene blue adsorption ability in a range of 50 ml/g or more, and (b)an iodine adsorption ability in a range of 750 mg/g or more.
 9. Acolored composition comprising the colorant preparation of claim
 8. 10.The colored composition according to claim 9, wherein the composition isselected from the group consisting of a food, a perfumery, a cosmetic, apharmaceutical, a quasi-drug, and a feedstuff.
 11. A gardenia bluehaving a total content of geniposide and genipin in a range of 300 ppmor less, which is a total content of geniposide and genipin when thegardenia blue has a color value of 100.